The present invention relates to protein Rim2, which is a novel isoform of Rim, i.e., a protein that interacts with a low molecular G protein Rab3 and is proposed to serve as a regulator of Rab3-dependent synaptic vesicle fusion, and which specifically interacts with the GDP/GTP exchange factor (GEFII; a cAMP sensor). More specifically, the present invention relates to elucidation of the mechanisms of intracellular vesicle transport and secretion, and to the novel protein Rim2 which is useful in diagnosis of endocrine-related diseases or neuropathy and in development of agents for prevention and treatment thereof, the gene encoding Rim2 and an antibody addressed to Rim2 protein.
Rim2 is considered to be a regulatory factor of vesicle fusion. It was found in the course of the present invention that the protein is expressed predominantly in endocrine tissues and endocrine- and neuroendocrine-derived cell lines. GTP-Rab3/GEFII/Rim complex is thought to participate in the regulation of exocytosis of neurons and endocrine cells, in a cAMP-dependent and protein kinase A (PKA) independent manner.
Transport of substances between cell organelles, which are unit membrane-enclosed structures such as endoplasmic reticulum, is conducted by intracellular vesicle transport. In endocrine cells including pancreatic xcex2-cells and pituitary cells, peptides/proteins synthesized at ribosomes are received by the endoplasmic reticulum, from which they are transported in vesicles, which are transformed into secretory vesicles through the Golgi body and transported to the cell membrane, where they are released out of the cell via a step which includes fusion of the membranes. In neurons, neurotransmitter-containing precursors of synaptic vesicles are formed in Golgi bodies and transported by microtubules along the axon and stored at the synapse. Depolarization of the pre-synoptic membrane causes the vesicles to fuse with the pre-synaptic membrane and thus the neurotransmitters are released. This type of secretion based on the fusion of the vesicles and the cell membrane is called exocytosis.
In contrast, when extracellular substances such as hormones including cell growth factors are bound to the cell membrane, the complexes thus formed are invaginated into the cell to form endosomes. This type of uptake of environmental substances is called endocytosis.
Formation of vesicles, such as by budding, commonly observed both in exocytosis and endocytosis, and docking and fusion, the phenomena observed in process of their transportation and binding to other membrane systems, are regulated by a GTP-binding, low-molecular protein, called G protein. More than 30 types of this protein are known. The group of the proteins, which are also classified in Rab family, regulate the intracellular vesicle transport system.
With regard to the intracellular vesicle transport system, it is understood today that a cell is in a resting state when Rab protein occurs in a bound form to guanine nucleotide diphosphate (GDP), and that budding, docking and fusion are triggered as a result of a process in which a protein having GEF activity act on Rab protein and converts it to GTP-binding Rab protein, to which GTP binds to form a GTP-Rab complex, which in turn binds to a corresponding target protein on the membrane.
Stimulus-secretion coupling plays an important role in exocytosis observed in many cell types including neurons and endocrine cells [J. E. Rothman, Nature 372:55(1994); T. C. Sudhof, Nature 375:645 (1995)]. While a rise in intracellular Ca2+ concentration is important in the regulation of exocytosis, other signals are also known to play important roles, cAMP (cyclic adenosine-3xe2x80x2,5xe2x80x2-monophosphate)/PKA (cAMP-dependent protein kinase A) signaling pathway is known to regulate exocytosis in many of neurons, neuroendocrine cells and endocrine cells. In particular, cAMP has been thought to mediate long-term potentiation by increasing neurotransmitter release in the brain [R. D. Hawkins et al. Ann. Rev. Neurosci. 16:625(1993); G. Lonart et al., Neuron 21:1141(1998)]. cAMP also regulates exocytosis responsible for insulin release from pancreatic xcex2-cells and amylase release from parotid acinar cells [P. M. Jones and S. J., Persaud, Endocrine. Rev. 19:429(1998); E. Renstrom, et al., J. Physiol. 502:105(1997); K. Yoshimura, Biochim. Biophys. Acta 1402:171(1998)].
In addition to its role in PKA-dependent phosphorylation of regulatory proteins associated with the process of exocytosis, it is known that cAMP also acts directly on the exocytotic machinery in neurons and non-neuronal cells [G. Lonart et al., Neuron 21:1141 (1998); E. Renstrom et al., J. Physiol. 502:105 (1997); K. Yoshimura, Biochim. Biophys. Acta, 1402:171(1998)].
During the search by the yeast two-hybrid screen (i.e., a method for detection of the interaction between two proteins in yeast cells) for an intracellular signaling molecule directly coupling to a sulphonylurea receptor, a component of pancreatic xcex2-cell ATP-sensitive K+ (KATP) channels [N. Inagaki et al. Proc. Natl. Acad. Sci. U.S.A. 91,2679 (1994)], a cAMP sensor protein (called xe2x80x9cCAMPSxe2x80x9d) was identified and it was found that the protein has two putative cAMP binding domains, a Pleckstrin homology domain (PH domain), and a guanine nucleotide exchange factor (GEF) homology domain.
In the course of this study, two study groups independently reported cAMP binding proteins that activate Rap1, a member of the small G binding proteins [J. de Rooiji et al. Nature 396:474 (1998); H. Kawasaki et al. Science 282:2275 (1998)], and CAMPS was incidentally revealed to be a mouse homologue of cAMP-GEFII [H. Kawasaki et al. Science 282:2275 (1998)].
Though the mechanisms of intracellular vesicle transport system have thus gradually been clarified, substantial part of them remains still unknown. Further progress is needed for the understanding of the mechanisms so as to provide diagnostic agents or therapeutics for a variety of diseases which involve neurons or endocrine cells.
Unlike the former suggestion that only a single cAMP binding domain was present in cAMP-GEFII, the study by the present inventors suggested the presence of two putative cAMP binding domains (cAMP-A and cAMP-B), based on a sequence alignment of cAMP-GEFII sequence and regulatory subunits of PKA. FIG. 1 shows the sequence alignment of the cAMP binding domains. The cAMP binding domains A and B (cAMP-A and cAMP-B, respectively) of cAMP-GEFII and the cAMP binding domains A and B of the PKA regulatory subunit Ixcex1 (RIxcex1-A and RIxcex1-B, respectively) are shown. The invariant residues in the different cAMP-binding domains are indicated by black boxes.
As shown in FIG. 2, a glutathione-S-transferase (GST)-cAMP-A fusion protein bound to [3H]cAMP with a dissociation constant (Kd) of, xcx9c10 xcexcM, while the binding of [3H]cAMP to a GST-cAMP-B fusion protein was not evident under the same conditions.
FIG. 2 shows the binding of cAMP to cAMP-A. GST-cAMP-A (filled circles) or GST-PKA RIxcex1 (open circles) was incubated with different concentrations of [3H]cAMP (0-50 xcexcM). The data for cAMP-A or PKA RIxcex1 are normalized relative to maximal cAMP binding activities. Kd values are 10.0xc2x12.3 xcexcM and 23.7xc2x10.6 nM for cAMP and PKA RIxcex1, respectively.
In the cAMP-B domain, the amino acid residue 423, which originally is glutamic acid (Glu), is substituted with lysine (Lys). This glutamic acid residue is important for CAMP binding. Considering that a more rapid dissociation than the wild-type was observed with a PKA regulatory subunit having an equivalent mutation (E-200-K), cAMP-B may also dissociate cAMP rapidly. Thus, a possibility remains that cAMP binds to the cAMP-B domain.
As identification of a target molecule of CAMPUS, cAMP-GEFII, would serve to show its physiological role, the present inventors attempted to find a molecule that interacts with cAMP-GEFII by means of a yeast two-hybrid screen (YTH) method on the MIN6 cDNA library (See xe2x80x9cIdentification of Interacting molecules by YTH Methodxe2x80x9d).
Surprisingly, the present inventors found that cAMP-GEFII interacts with a novel isoform (named xe2x80x9cRim2xe2x80x9d by the present inventors) of Rim (a molecule which specifically interacts with Rab3: Rab3-interacting molecule: Hereinafter referred to as xe2x80x9cRim1xe2x80x9d). Rim1 protein is a putative effector of the small G protein Rab3 and is proposed to serve as a Rab3-dependent regulator of synaptic vesicle fusion [Y. Wang et al. Nature 388:593(1997)].
The full-length novel protein Rim2 sequenced by the present inventors, which consists of 1590 amino acid residues, was found to have 61.6% identity with rat Rim1. As FIG. 3 shows, a zinc finger, PDZ and two C2 domains were found highly conserved between Rim1 and Rim2.
Based on the above findings, the present invention provides a protein having the amino acid sequence set forth under SEQ ID NO:1 in the Sequence Listing.
The present invention further provides a protein having an amino acid sequence with one or more amino acids deleted, substituted, inserted or added relative to the amino acid sequence set forth under SEQ ID NO:1 in the Sequence Listing and which has a property to interact with GDP/GTP exchange factor II.
The present invention further provides a mouse gene which encodes the following proteins (1) or (2):
(1) a protein having the amino acid sequence set forth under SEQ ID NO: 1 in the Sequence Listing,
(2) a protein having an amino acid sequence with one or more amino acids deleted, substituted, inserted or added relative to the above-identified amino acid sequence and which has a property to interact with GDP/GTP exchange factor II.
In the present specification, xe2x80x9cone or morexe2x80x9d amino acid residues are generally several (e.g., 3 or 4) to 10 residues.
The present invention further provides a DNA having a nucleotide sequence set forth under SEQ ID NO:2 in the Sequence Listing, the DNA being a cDNA corresponding to the above protein having the amino acid sequence set forth under SEQ ID NO:1 in the Sequence Listing.
The present invention further provides a DNA having a nucleotide sequence with one or more nucleotides deleted, substituted, inserted or added relative to the nucleotide sequence set forth under SEQ ID NO:2 in the Sequence Listing and encoding any one of the above proteins. Herein, xe2x80x9cone or morexe2x80x9d nucleotides are generally several (e.g., 3 or 4) to 10 nucleotides. A variety of such nucleotide sequences with one or more nucleotides deleted, substituted, inserted or added can be readily prepared by those skilled in the art by making use of the familiar knowledge on degeneracy of the genetic code.
The present invention further provides a DNA having the nucleotide sequence of the coding region of the any one of the above DNA""s or of a DNA having the nucleotide sequence set forth under SEQ ID NO:2 in the Sequence Listing.
The present invention further provides a DNA fragment consisting of a part of any one of the above DNA""s.
The present invention further provides a probe comprising a DNA which hybridizes with the DNA consisting of any one of the above nucleotide sequences.
The present invention further provides a primer DNA fragment consisting of a partial sequence of any one of the above nucleotide sequences.
The present invention further provides a recombinant vector having any one of the above DNA""s.
The present invention further provides a monoclonal or polyclonal antibody directed to any one of the above proteins.
The present invention further provides a diagnostic agent for human use comprising any one of the above probes or antibodies. The diagnostic agent is useful in the test for such diseases as secretion disorders in secretory systems including pituitary, hypothalamus, pancreatic xcex2-cells and parotid gland, or the test for brain-nervous system diseases.
The present invention further provides a therapeutic agent for any one of the above diseases.